1. Field of the Invention
The present invention relates to the detection of urobilinogen in a test sample. More particularly, it relates to an improved test composition, device and method for detecting urobilinogen.
2. Description of the Prior Art
The analysis of urobilinogen species in urine is an old art which had its beginning around the turn of the century (1901) when Ehrlich discovered that p-dimethylaminobenzaldehyde reacts with urobilinogen in the presence of strong acid such as HCl to give a red color. This reaction was concurrently discovered by Proescher in the same year, and hence, the test is interchangeably referred to as the "Ehrlich" or "Ehrlich-Proescher" reaction. The chemistry underlying these simultaneous discoveries still forms the basis of today's urobilinogen testing.
This extensive utility and wide acceptance in medical diagnostics notwithstanding, it was soon found that the basic Ehrlich reaction is susceptible to various interfering substances in urine. Specifically, it was found that indole and skatole derivatives, pyrrole compounds, sulfonamides, and other substances interfered with the reaction by generating the red color typical of a positive test result. In 1925 Terwen found that by adding sodium acetate not only was the urobilinogenaldehyde color intensified, but also the color due to indole and skatole derivatives was inhibited. Modifications of this improvement are still in use as evidenced by U.S. Pat. No. 3,447,905. More recently, attempts to provide additional diagnostic agents for use in urobilinogen detection have resulted in systems such as that disclosed in U.S. Pat. No. 3,630,680. To date, the significant urobilinogen tests which have been developed rely almost exclusively on the use of p-dialkylaminobenzaldehyde indicators such as p-dimethylaminobenzaldehyde. Such a product is currently marketed by the Ames Division of Miles Laboratories, Inc. as UROBILISTIX.RTM. reagent strips.
The present invention departs from the state of the art in that it is directed to a unique improvement in the Ehrlich reaction. The new composition provides shorter reaction time, more intense color formation and less interference by such urine constituents as nitrite, isonicotinic acid hydrazide, p-aminosalicylic acid and indole.
Moreover, the use of the presently claimed composition in preparing test devices for measuring urobilinogen results in a greatly simplified procedure. Prior to the present invention, so-called "dip-and-read" reagent strips for urobilinogen were prepared by a two-dip process. Thus, a first reagent solution was prepared by dissolving aminoacetic acid and fluoboric acid in an alcoholic water solvent. This first solution was used to impregnate a carrier matrix, for example paper, which was then dried. A second reagent solution was then prepared containing a stannic chloride dioxane complex and p-dimethylaminobenzaldehyde in methanol. The dried impregnated paper was then immersed into this second solution and dried.
Surprisingly, because of the resultant enhanced stability of the presently claimed reagent system, test devices can be prepared using only a single impregnation or one-dip method. Because of the unexpected stability of the present composition, all of the ingredients can be dissolved in a single reagent solution, thus eliminating the costly, cumbersome second dip.
Another advantage of the present invention is that it eliminates the need for the highly acidic components of prior art devices which caused degradation of paper carrier matrices. The present state of the art requires the use of such compounds as SnCl.sub.4 dioxane complex as a source of HCl in the reaction. Because of the relatively high reactivity of this component with paper in the presence of water, the two-dip preparation is necessary, the first dip to properly buffer and prepare the carrier matrix for the acid-producing complex. The present invention obviates the necessity of such a first step. The finished product utilizing the present composition enables a faster reaction time with the development of a much more intense color (yellow to deep red) than was heretofore available. In addition, the color range that is developed in the present invention is broader than that of the prior art, thereby enabling the use of more color blocks than prior art techniques over an equivalent range of urobilinogen levels.
To summarize the advantages of the present invention over the prior art, the rate of reaction is enhanced so that the waiting period for color development or other detectable response is greatly diminished; the color formed in the reaction is of much greater intensity than that of the prior art, thus facilitating greater sensitivity and accuracy; and a one-dip process can be used to manufacture the device as opposed to the two-dip process required in manufacturing the prior art reagent strips.